Fig 2: Fe65 Ser289 does not regulate Fe65-APP transcriptional activity.(A) HEK293 cells were transfected with the indicated plasmids for 24h prior to cell lysis and measurement of both firefly and renilla luciferase activities. To normalise for transfection efficiency the ratio of firefly/renilla luciferase values were calculated (each sample in triplicate). (B) The cellular samples used for the luciferase assay in (A) were analysed by Western blotting to confirm equal expression of both FLAG-Fe65 and FLAG-Fe65 C652F. (C) and (D) HEK293 cells were transfected with the indicated plasmids for 24h prior to luciferase assays and Western blot analysis as in (A) and (B). Data is the mean and standard deviation of triplicate samples and is representative of at least three independent experiments. The significance of the observed changes in relative luciferase activity were investigated using a paired t-test (two-tailed). Levels of significance were defined as follows: p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).

Fig 3: NaR enhances microglial phagocytosis of Aβ in vivo and in vitro.(A) Representative images of Aβ (6E10) plaques and microglia (Iba1) costaining in the cortex from APP/PS1 and NaR-treated APP/PS1 mice. (B) Quantification of microglial cells within 20 μm of the plaque surface (n = 107 to 144 plaques per group). (C) Plaques were divided into small, medium, and large according to their size, and the number of microglia per plaque was quantified. (D) Representative images of Aβ (6E10) plaques, microglia (Iba1), and phagosome (CD68) costaining in the cortex from APP/PS1 and NaR-treated APP/PS1 mice. (E and F) Quantification of the percentage of phagosome area and internalized Aβ using ImageJ Pro Plus software (n = 10 per group). (G) Confocal analysis of microglial phagocytosis of FITC-Aβ42 in the presence or absence of NaR after uptake for 4 hours in cultured primary microglia; CytoD served as a negative control. (H) Quantification of internalized FITC-Aβ42 using ImageJ software (n = 48 to 60 per group). a.u., arbitrary units. (I) FITC-Aβ42 uptake index in the presence or absence of NaR after uptake for the indicated time in cultured primary microglia (n = 6 per group). Data are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test.

Fig 4: CX-4945 inhibits DYRK1A-mediated APP and PS1 phosphorylation in mammalian cells. 293T cells were transfected with the indicated plasmids expressing APP (A), PS1 (C) and/or DYRK1A. Transfected cells were treated with the indicated doses of CX-4945 for 6 h, and then total cell extracts were subjected to western blot analysis. The phosphorylated and total APP and PS1 proteins from western blotting were quantified, and the amounts of protein relative to those of DMSO-treated samples are shown in B and D, respectively. The mean±s.d. values were determined from two independent assays. p-APP, phosphorylated APP; p-PS1, phosphorylated PS1; Vec, empty vector. / denotes the ratio of the two indicated proteins.

Fig 5: Supplementation with CPPs ameliorated Aβ pathology in N2a-APP cells. (A)The expression of APP in N2a-APP cells was estimated by western blot compared with N2a cells(n=3). (B) Quantification of the western blot results with APP normalized to GAPDH. N2a-APP cells were treated with CPPs at the indicated concentrations of 50, 100, 200 μg/ml for 24 h. (C) N2a-APP cells were assayed for cell viability, n = 3. (D) ELISA assay for Aβ1-42 levels, n = 3. (E–H) The expression of APPα, APPβ and BACE1 proteins in the N2a-APP cells was measured by Western blotting, normalized to GAPDH, n = 3. (I) Relative BACE1 activity was detected in the cells (n=6). P value significance is calculated from a one-way ANOVA or Student’s t-test. *P < 0.05, **P < 0.01 vs. cells without CPPs treatment.